Many organic reagents form color precipitates with metal ions. The first report, in 1884, concerned the use of 1-nitroso-2-naphthol, as a precipitant for cobalt and other ions. Since then, colored ion reactions have played a key role in inorganic chemical analysis. A particularly intense color is associated with the formation of inner complex compounds, which are salts of metal atoms which manifest auxiliary valences towards certain atoms of a chelating acid radical. Nitrosophenylhydroxylamine, also known as cupferron, was one of the first organic reagents to be used for quantitative analysis which relied on the formation of inner complex salts. As illustrated by copper glycinate, copper salts can also form inner complex compounds. Many distinctive properties of metals not observed in normal salts appear upon formation of inner complex salts. Normally unstable compounds can be stabilized through inner complex formation. Inner complex compounds are especially important as analytical reagents, due to the formation of principal and auxiliary valence rings which frequently accompany anomalous solubility and/or tinctorial qualities. Feigl, F., Chemistry of Specific, Selective and Sensitive Reactions, Academic Press, N.Y. (1949).
Catalytic activity is exhibited by metals that can easily and reversibly change from one to another of several valences. Complex compounds often contain metals with valences which either do not occur in normal salts or which allow stable compounds to be formed with some unusual reagents. The remarkable discovery that copper (II) salts can catalyze reductions as well as oxidations is explained by alterations of valence to the unstable and strongly reducing Cu(I) ion, and to the unstable and strongly oxidizing Cu(III) ion. Feigl, supra.
A large variety of means for detecting copper, often at extreme dilutions, are based on the wide range of reactivity of copper ions. Hodgman, C. D. et al., Handbook of Chemistry and Physics, Chemical Rubber Publishing Co., Cleveland, Oh. (1962). Cavallini, D. et al., Arch. Biochem. Biophys. 124:18-26 (1968). Reagents employed in these assays include benzidine blue, salicylaldehyde and its oxime, mixtures of benzidine acetate and potassium bromide, cyanide, 8-hydroxyquinoline, phenylglycine, alpha amino-n-caproic acid and other amino acids, anthranilic acid, dicyandiamidine, 4-hydroxybenzothiazole, acyloinoximes, quinoline-8-carboxylic acid, precipitates such as rubeanates, iodine, and finally alpha-alpha'-alpha-tripyridyl. Partrington, J. R., Textbook of Inorganic Chemistry, 5th Ed., MacMillan and Co., London (1946). Feigl, F., supra.
Despite the extraordinary reactivity of copper ions with a wide variety of reagents, the use of copper as a reagent to test for other substances has been relatively limited. Cupric and cuprous salts have been utilized to detect mercaptobenzothiozole, sulfite, thiocyanate, xanthate, and thionalid. They have also been used as a reagent in gas analysis for carbon monoxide, ammonia, cyanide, and acetylene. Feigl, F., supra: Hodgman, supra: and Partrington, supra. The most conspicuous use of copper salts as analytical agents has been for the analysis of glucose and other sugars. At least eight chemical tests for sugars, some of them in tartrated media, have been published. Hodgman, C. D. et al., supra. These tests include Fehling's, Bang's, Barfoed's, Benedict's, Bertrand's, Hagedorm and Jensen's, Somogyi's, Nylander's, and Munsen and Walker's tests. Most of these tests utilize CuSO.sub.4 as the primary reagent. However, these tests are especially laborious and require one or more heat steps followed by titration with a final reagent. Hodgman, C. D., supra.
Tartrated copper salts have also been utilized for years in the Lowry protein assay. Hodgman, C.D., supra. Despite the knowledge that a number of substances tend to cross-react in these assays, no effort has been made to exploit these other potentially specific reactions.
Traditional assays often require separate tests for individual analytes, which further require a number of steps and require spectrophotometric or other, sophisticated detection methods. For example, DTT is assayed by reaction with Ellman's reagent. Ellman, G. L., Arch. Biochem. Biophys. 82:70-77 (1959). McCloud, R. W., Anal. Biochem. 112:278-281 (1981). Cysteine may be assayed by spectrophotometric titration with ferricyanide which oxidizes cysteine to cystine, or by PCMB, o-iodosobenzoate, silver ion methods, or by monitoring O.sub.2 consumption or H.sub.2 O.sub.2 generation by cysteine-metal ion complexes. Cavallini, supra; Michaelis, L. et al., J Biol. Chem. 83:191-210 (1929). In addition, cystine and cysteine have been assayed using a uric acid reagent and sodium sulfite. Folin, O. et al., J. Biol. Chem. 83:103-108 (1929). Glycerol is usually determined by a two-step enzymatic assay. Bergmeyer, H.-U., Methods of Enzymatic Analysis, Academic Press, New York, N.Y. (1963). It would be desirable to have a test that allows for the simultaneous detection of all these analytes by a simple color test.